The herein disclosed invention finds applicability in the field of cell culture, as well as in the field of tissue substitute for tissue replacement.
Attempts at replacing or rebuilding diseased or damaged structures in the human body go back to 3000 B.C. It was not until the middle of the 1900""s, however, that the use of synthetic materials for rebuilding body structures met with widespread and reproducible success. Advances in material science and biomaterials and science have afforded much of the success. The need for new and better implants exists in every field of medicine in which disease or trauma can be treated surgically.
As technology advances continue to improve the state of the art, the standards for successful implants continue to improve including strength, biocompatibility and elasticity. The new research being conducted today on growth factors and controlled drug release tell of the day when implant material will be expected to promote healing, dissipate disease and stimulate tissue regeneration. New horizons for implants and implantable materials are being developed through the coordinated efforts of biomedical engineers and surgeons who are best positioned to appreciate the problems that patients are facing and that need to be addressed.
Tissue engineering has opened a new frontier in the development of implant materials for surgery. The surgical specialties which are more apt to deal with cartilage grafts substitutes and bone graft substitutes are the fields of orthopedics, otolaryngology-head and neck surgery and facial plastic reconstructive surgery. Cartilage and bone graft substitutes can be used as temporary scaffolds in which new cartilage and/or new bone may grow, encouraging tissue regeneration rather than inert tissue replacement. The regeneration of tissue in a controlled and predictable manner may be achieved with biodegradable implants, where the scaffold or template will degrade and be absorbed by the body, and the living cartilage or bone cells will remain. Implants such as these will eliminate the concerns about the long-term stability and safety of implant material currently used for tissue augmentation, and obviate the concern about long-term implant failure due to mismatch at the implant-tissue interface.
Articular cartilage is frequently damaged in the course of common activities. In addition, osteoarthritis leads to the initial focal wear of articular cartilage in many joints, particularly the hip and the knee. There are approximately 500,000 total hip and knee prostheses implanted in the United States every year and more than 1,000,000 arthroscopies of the knee. In the case of the replacements, the condition usually started with focal loss of articular cartilage. Under normal circumstances, articular cartilage, once damaged, does not heal. Current treatment methods consist mostly of alleviating symptoms through the use of activity modification (restrictions), weight-bearing protection (canes, crutches, walkers), analgesics and anti-inflammatory drugs. Recently, the FDA has approved autologous cell therapy. In this method, Chondrocytes are harvested arthroscopically, multiplied in tissue culture and implanted in the articular cartilage defect in the knee under a periosteal flap. The procedure costs $30,000. Alternative concepts are in development, but none are approved.
The inventors have developed a unique way of multiplying human chondrocytes so that they rapidly multiply and don""t lose their phenotypic expression. They invest these cells in a scaffold that allows the interaction of the scaffold and the cells to reproduce the physical attributes of the cartilage the implant is intended to replace. No current therapy other than cadaver cartilage transplant offers this potential. Cadaver cartilage transplant is ineffective over the long term, is expensive and transplant material is not readily available. It must be emphasized that neither the cells nor the scaffold alone will work. In both instances, success is dependent on an uncontrolled response from the body in an uncontrolled environment. In the past, such response has been unpredictable and the development of such a response has required long periods of inactivity on the part of the patient. The approach provides a mature mechanically functional implant to the patient at the time of implantation, requiring only sufficient time for the implant to unite to the host (4-6 weeks).
Most of the patients undergoing arthroplasty for the knee started with a focal lesion of the a knee that would have been amenable to treatment with chondrocyte-implant. At least 50% of the patients undergoing arthroscopy of the knee have a focal articular cartilage lesion that is amenable to treatment with the chondroctye implant.
The ideal implant material for the future will be capable of forming a living bond to tissue, providing a scaffold or template to allow cells to grow and multiply and lay a matrix that will fill defects in the body and promote tissue healing. The field of this invention involves the use of polymer constructs as scaffolds or templates to allow the growth and multiplication of autologous cells in culture, including chondrocytes (cartilage cells), oesteocytes (bone cells) oesteoblasts, chondrogenic cells, pluripotential cells and mucosal cells for tissue replacement and/or coverage.
Chondrocytes are the sole cellular component of articular cartilage. They are sparsely distributed in the cartilaginous matrix and occupy less than 5% of the tissue volume. Chondrocytes produce and break down macromolecules that make up cartilage. These macromolecules consist primarily of a collagen type II network embedded with high-molecular-weight proteoglycans. By maintaining the integrity of articular cartilage, chondrocytes play a key role in the load-bearing function of the joint.
Several strategies have been explored to expand the number of chondrocytes ex vivo. Most of these attempts involve propagating cells in monolayer culture, which allows them to proliferate. However, chondrocytes propagated in monolayer culture lose their original characteristics by assuming a fibroblastoid morphology and shift from production of collagen type II to type I. They also change synthesis of high- to lower-molecular-weight proteoglycans.
Attempts to maintain the original phenotype in monolayer culture have had some success by seeding chondrocytes in high density. Despite these modifications in culture conditions, not all chondrocytes reverted to the spherical, collagen type II producing cells. Under culture conditions in which cells are restrained from spreading out as in monolayer culture, chondrocytes were inhibited from proliferating. These methods are not able to provide sufficient numbers of chondrocytes with unaltered phenotype needed for analysis. There is therefore a need for a culture system that would allow chondrocytes to proliferate, as well as maintain their original characteristics.
One culture system that has been shown to facilitate cell proliferation, as well as maintain synthesis of cellular products, is the microcarrier suspension culture. This culture system was based on the original xe2x80x98bioreactorxe2x80x99 design to grow bacteria in large quantities.
Microcarriers have been utilized in the prior art to grow anchorage-dependent mammalian cells in large quantities. Cells attached onto microcarriers are maintained in suspension spinner culture under controlled levels of pH, oxygen, nutrient supply and mechanical agitation. At an appropriate time the cells are harvested. Existing methods provide fairly simple procedures for obtaining secreted products from the spent culture. However, the ease of recovering high yields of viable and functional cells from microcarriers has been a problem. Several factors contribute to the problem of harvesting viable cells. These factors include the chemical composition of the microcarriers, the charge on the microcarrier surface and the method of cell harvesting. The most common method of cell harvesting used is by trypsinizaion, which may not completely retrieve cells from the microcarriers.
With all of this in mind, applicants have perfected means for cell culture, retrieval and methods for using the cells for tissue implants when used along with polymer constructs.
U.S. Pat. No. 4,994,388 to Hillegas et al discloses a method for culturing and harvesting, anchorage-dependent cells employing insoluble microcarrier beads coated with collagen. The cells attach and grow on the collagen coated microcarrier. Once growth is complete, the collagen is digested off of the microcarrier, and the cultured cells are separated from the insoluble microcarrier. This patent also discloses that the patentees have, in contrast to using collagen coated beads, used beads made entirely of collagen and found these latter beads to support the growth of some cells very well, but that other cells did not proliferate well on collagen-only beads. The patentees also found in contrast to collagen coated beads that collagen-only beads present a slower attachment of cells to collagen. In addition, the patentees found that harvesting cells from collagen-only beads presented difficulties in separation of the cells from the media in that there was contamination of the cells by soluble fragments of collagen. While the patent discloses applicability of their invention to anchorage-dependant cells, the patent does not disclose any specific cell, nor are chondrocytes specifically disclosed. The disclosure of the patent to Hillegas et al has no specific embodiment of a process for carrying out separation of a specific cell from a specific substrate with a specific enzyme.
Mundt in U.S. Pat. No. 5,100,799 discloses a method for releasing cells from microcarriers using trypsin to release the cells. The composition of the microcarrier is not disclosed in this patent.
Kandel in U.S. Pat. No. 5,326,357 discloses cartilage tissue reconstituted on a substrate. The method of this patent involves reconstituting cartilage tissue in vitro from chondrocytes cultured on a substrate.
Green in U.S. Pat. No. 4,304,866 teaches a method for producing a transplantable sheet of living keratinous tissue by culturing keratinocytes in a culture vessel and subsequently enzymatically detaching a sheet of keratinous tissue employing an enzyme. These sheets of keratinous tissue are to be used for producing skin grafts.
Civin in U.S. Pat. No. 5,081,030 teaches the release of cells from an affinity matrix. The enzyme used by Civin can be an enzyme selected from the group consisting of carbohydrases, proteases and lipases. The carbohydrase is to be used for cell surface glycolipids and glycoproteins. Cell culture on a microcarrier beadlet is not disclosed in this patent.
An object of this invention is to efficiently propagate cells in vitro on the surface of microcarrier spheres or microcarrier beadlets.
A further object of this invention is to propagate cells on microcarriers to promote proliferation of the cells and also to continue the synthesis of cell by-products characteristic of the cells.
A most important object of this invention is to produce cells free of microcarriers without the need to physically separate the carrier from the cell.
A still further object is to be able to retrieve cells for testing or analysis.
A further important object of this invention is to produce viable cells by culture for further propagation in tissue culture.
An important object of this invention is to efficiently culture cells in vitro to be subsequently used in vivo.
A significant object of this invention is to prepare cells in culture to be used in animal or human transplantation.
A major object of this invention is to produce cells, which are useful for forming an implant device.
A main object of the invention is to prepare a polymer having medical applicability.
Another object of this invention is to prepare polymers useful for supporting the growth of cells or tissues.
An important object of this invention is to develop a polymer to be used for preparing scaffolds for cells.
An important object of this invention is to form polymer beads used as a substrate for culturing attached cells, as well as a substrate for preparing cell seeded scaffold implants.